With the recent dramatic rise of antibiotic-resistant pathogens and infectious diseases, the need for new antimicrobial agents is urgent (Cohen et al., 1992, Science 257:1050-1055). For example, recently strains of Enterococcus faecium that are resistant to vancomycin have been observed (Moellering, 1990, Clin. Microbiol. Rev. 3:46-65). As vancomycin is considered to be the antibiotic of last resort for several pathogens, strains resistant to vancomycin pose a serious health threat to society. Despite this urgency, in more than ten years only one completely different type of antibiotic, a streptogramin mixture called Synercid (Rhone-Poulenc Rorer, Collegeville, Pa.), has reached Phase III clinical trials (Pfeiffer, 1996, "New Anti-Microbial Therapies Described," Genetic Engineering News 16(8):1).
Recently, a new class of antimicrobial or antibiotic agents based on naturally-occurring antimicrobial peptides produced within plants, animals and insects have been discovered. These peptides include, among others, cecropins (Hultmark et al., 1980, Eur. J. Biochem. 106:7-16; Hultmark et al., 1982, Eur. J. Biochem. 127:207-217), apidaecins (Casteels et al., 1989, EMBO J. 8:2387-2391), magainins (Zasloff, 1987, Proc. Natl. Acad. Sci. U.S.A. 84:5449-5453; Zasloff et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:910-913), tachyplesins and analogues of tachyplesins such as polyphemusins (Nakamura et al., 1988, J. Biol. Chem. 263:16709-16713; Miyata et al., 1989, J. Biochem. 106:663-668), defensins (Lehrer et al., 1991, Cell 64:229-230; Lehrer et al., 1993, Ann. Rev. Immunol. 11:105-128; U.S. Pat. No. 4,705,777; U.S. Pat. No. 4,659,692; U.S. Pat. No. 4,543,252), .beta.-defensins (Selsted et al., 1993, J. Biol. Chem. 288:6641-6648; Diamond et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:3952-3958), insect defensins (Lambert et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 88:262-265; Matsuyama and Natori, 1988, J. Biol. Chem. 263:17112-17116), and protegrins (Kokryakov et al., 1993, FEBS 337:231-236; Zhao et al., 1994, FEBS Letters 346:285-288; Migorodskaya et al., 1993, FEBS 330:339-342; Storici et al., 1993, Biochem. Biophys. Res. Commun. 196:1363-1367; Zhao et al., 1994, FEBS Lett. 346:285-288; Manzoni et al., 1996, FEBS Lett. 383:93-98; U.S. Pat. No. 5,464,823). The discovery of these new classes of antimicrobial peptides offers hope that some might be developed into agents that can be used against microorganisms of medicinal importance. Those of animal origin are of particular importance, as these antimicrobial peptides generally exhibit activity against antibiotic-resistant bacterial strains and have a lower frequency of resistance as compared to conventional antibiotics (Steinberg et al., 1996, "Protegrins: Fast Acting Bactericidal Peptides," presented at: Intl. Symposium on Staphylococci and Staphylococcus Infections, Aix les Bains, France). At least one of these peptides, magainin MSI-78, is currently in Phase III clinical trials for infections associated with diabetic foot ulcers (Craig, Aug. 17, 1995, BioWorld Today 6(158):1).
The use of peptides as therapeutic agents in general, however, has not been completely satisfactory. Peptides composed of L-amino acids undergo rapid proteolysis in the gut, making oral administration, the method generally associated with the highest patient compliance, extremely difficult. Additionally, peptides degrade fairly rapidly in serum and therefore must be administered in large doses which often can cause numerous adverse side effects and serious toxicity. As peptides are expensive to manufacture, high dosage levels contribute significantly to the overall cost of peptide therapeutics. Furthermore, the flexibility of the peptide structure in solution is often associated with low biological activity and/or selectivity.
It has now been discovered that cyclic peptides related to the tachyplesin and protegrin classes of antimicrobial peptides exhibit broad spectrum antimicrobial activity typically associated with these classes of peptides while overcoming many of the disadvantages of peptide therapeutics discussed above. Thus, the cyclic peptides are ideally suited for use as antimicrobial therapeutic agents. For example, cyclic peptides are more resistant to proteolytic degradation and therefore have a greater potential for oral administration and/or lower dosage levels than non-cyclized peptides. Cyclization also confers structural stability without interfering with the side chains necessary for bioactivity, potentially leading to greater efficacy.
Cyclic analogues of the various known classes of antimicrobial peptides have not been reported in the literature. The only cyclic antibiotic peptide described in the literature are gramicidin-S (Tamaki et al., 1995, Int. J. Peptide Protein Res. 45:299-302; Gause, 1994, Lancet 247:715; Battersby et al., 1951, J. Am. Chem. Soc. 73:1887; Schwyzen et al., 1957, Helv. Chim. Acta 40:624), grastatin (Zharikova et al., 1972, Vestn. Mosk. Univ. Biol. Pochivoved 27:110; Myaskovskaya et al., 1973, Vestn. Mosk. Univ. Biol. Pochivoved 28:123) and tyrocidines (Laiken et al., 1969, J. Biol. Chem. 244:4454; Gibbons et al., 1975, Biochemistry 14:420). Significantly, these cyclic peptides are known to be toxic and do not contain positively-charged amino acid residues in loop or turn regions of the molecules--a feature of the novel class of cyclic peptides described herein thought to be important for broad-spectrum antimicrobial activity and improved efficacy towards antibiotic-resistant microbes.